Background: High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays.

Results: The mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 10(6) cells/ml, 93.3%) were compatible to those obtained with Ficoll (1.34 × 10(6) cells/ml, 97.2%). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles.

Conclusions: Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells' handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations.

Fig2: Yield and viability of PBMC isolated by the Ficoll and CPT protocols. a The mean numbers of PBMC per ml of blood obtained by Ficoll or CPT isolation procedure from 6 healthy donors. b The mean viability of PBMC freshly isolated from the same 6 healthy donors by either Ficoll gradient separation or CPT technique. No significant differences (P < 0.05) in PBMC yield or viability between the two isolation protocols were found. Error bars indicate standard error of the mean

Mentions:
The number and viability of PBMC from 6 healthy donors was assessed immediately following isolation by either the Ficoll or CPT technique and compared between the two. As described in the Methods, a RBC lysis step was included after purification of PBMC by both Ficoll and CPT protocols to minimize the impact of potentially contaminating RBC- and reticulocyte-derived RNA on downstream RNA and gene expression analyses. To correct for variation in the volume of blood used for PBMC isolation, a relative PBMC yield was calculated by dividing the total number of cells by the milliliters of blood from which the cells were isolated. The mean number of PBMC per ml of blood isolated by using the Ficoll method was 1.16 × 106 cells/ml (SEM = 1.49 × 105, range = 5.71 × 105 – 1.67 × 106) compared to 1.34 × 106 cells/ml (SEM = 1.19 × 105, range = 9.43 × 105 – 1.71 × 106) for the CPT technique (Fig. 2a). Thus, there was no significant difference in the number of PBMC isolated between the two methods (P = 0.398). The mean number of PBMC obtained per each CPT isolation (9.87 × 106, SD = 3.71 × 106, range = 4.95 × 106 – 1.80 × 107; n = 29) was comparable to that reported by the manufacturer (1.27 × 107, SD = 4.64 × 106, range = 7.02 × 106 – 2.14 × 107; n = 10). Both methods also resulted in isolation of similarly highly viable PBMC. The mean viability of Ficoll-isolated PBMC was 97.2 %, while that obtained by CPT method was 93.3 % (Fig. 2b) and this was not significantly different (P = 0.057).Fig. 2

Fig2: Yield and viability of PBMC isolated by the Ficoll and CPT protocols. a The mean numbers of PBMC per ml of blood obtained by Ficoll or CPT isolation procedure from 6 healthy donors. b The mean viability of PBMC freshly isolated from the same 6 healthy donors by either Ficoll gradient separation or CPT technique. No significant differences (P < 0.05) in PBMC yield or viability between the two isolation protocols were found. Error bars indicate standard error of the mean

Mentions:
The number and viability of PBMC from 6 healthy donors was assessed immediately following isolation by either the Ficoll or CPT technique and compared between the two. As described in the Methods, a RBC lysis step was included after purification of PBMC by both Ficoll and CPT protocols to minimize the impact of potentially contaminating RBC- and reticulocyte-derived RNA on downstream RNA and gene expression analyses. To correct for variation in the volume of blood used for PBMC isolation, a relative PBMC yield was calculated by dividing the total number of cells by the milliliters of blood from which the cells were isolated. The mean number of PBMC per ml of blood isolated by using the Ficoll method was 1.16 × 106 cells/ml (SEM = 1.49 × 105, range = 5.71 × 105 – 1.67 × 106) compared to 1.34 × 106 cells/ml (SEM = 1.19 × 105, range = 9.43 × 105 – 1.71 × 106) for the CPT technique (Fig. 2a). Thus, there was no significant difference in the number of PBMC isolated between the two methods (P = 0.398). The mean number of PBMC obtained per each CPT isolation (9.87 × 106, SD = 3.71 × 106, range = 4.95 × 106 – 1.80 × 107; n = 29) was comparable to that reported by the manufacturer (1.27 × 107, SD = 4.64 × 106, range = 7.02 × 106 – 2.14 × 107; n = 10). Both methods also resulted in isolation of similarly highly viable PBMC. The mean viability of Ficoll-isolated PBMC was 97.2 %, while that obtained by CPT method was 93.3 % (Fig. 2b) and this was not significantly different (P = 0.057).Fig. 2

Bottom Line:
High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology.No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found.Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations.

Background: High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays.

Results: The mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 10(6) cells/ml, 93.3%) were compatible to those obtained with Ficoll (1.34 × 10(6) cells/ml, 97.2%). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles.

Conclusions: Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells' handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations.